Detection and quantification of one or more target analytes in a sample using spatially localized analyte reproduction

ABSTRACT

The presence or absence of one or more target microbial analytes in a substance, such as a biological or environmental substance, is assayed by inoculating a growth medium with a sample of the substance. The medium may be combined with a labeled analyte-specific material (LASM) which can migrate through the substrate and which is homogeneously distributed throughout the medium. The LASM may be premixed with the medium, or may be added to the medium after inoculation with the substance. The nature of the medium is such that it will support target analyte reproduction so as to form target analyte colonies in or on the medium, and it will not allow the target analyte colonies to migrate on or within the medium. After the sample to be assayed is added to the medium, growth of the target analyte colonies in the sample will bind increasing quantities of the LASM, thereby creating localized intensely labeled areas in the medium which can be visually or photometrically detected. As the target analyte colonies grow on or in the medium they attract increasing quantities of the LASM which diffuses to the colonies through the medium causing the local target analyte colonies to become intensely labeled, which renders the colonies readily detectable. Each of the intensely labeled target analyte colonies in the medium may also be surrounded by areas of lower intensity due to local depletion of the LASM in the regions of colony growth. The number of intensely labeled target analyte colonies in the medium will be proportional to the concentration of the target analyte present in the sample. In the event that the target analyte is absent from the sample, then there will be no localized labeled microbe colonies in the medium.

TECHNICAL FIELD

This invention relates to a method and paraphernalia for visually orphotometrically detecting the presence or the absence of one or moremicrobial target analytes in a material sample; and enumerating theanalyte(s) when found to be present in the sample. The material beinganalyzed can be a biological material; an environmental material; a foodstuff material; or some other material which can harbor the targetanalyte(s). The target analyte(s) is (are) one or more specific livingorganisms such as microbes, bacteria, fungi, mycoplasma or protozoawhich may be found in the material being tested; and the presence orabsence of the target analyte or analytes is determined by causingmetabolic reproduction of the target analyte or analytes in a suitablegrowth medium. The analysis can be performed in a gel or a semi-solidgrowth material which supports selective growth of the targetanalyte(s), but restricts growth of other viable organisms which may bein the sample. In this manner, detectable target analyte colonies areformed in the medium. The colonies may be detected and counted byvarious techniques, including the use of visually or photometricallydetectable materials which will selectively bind to some portion of thetarget analyte(s).

BACKGROUND ART

The analysis of specimens for the presence or absence of target bacteriaand other viable organisms such as fungi and protozoa, wherein thespecimen is placed on a sterile growth medium, is well known. Variousbacteria are usually differentiated by their appearance, their chemicalreaction with growth or other medium, or by applying an enzymatic orimmunological reactant. Since bacterial colonies of different speciesappear similar, it is usually necessary to perform a number of tests ona number of colonies before one can be establish a correctidentification. This is especially a problem if it is necessary todistinguish bacterial subspecies, for example, finding the location of acolony of E. coli serotype 0157:H7 within a mix of other, apparentlyidentical E. coli colonies. There are growth medium with specializedcharacteristics which help in the rapid visual location of suchorganisms, but the more closely related the target organism is to thebackground, the more difficult it is to distinguish them on chemicaltests alone. It would be highly desirable if such colonies could berapidly and specifically located, particularly if no additionaloperations were required by the technician.

DISCLOSURE OF THE INVENTION

This invention relates to a method and paraphernalia for detecting thepresence or absence of one or more target microbial analytes in asample. Detection of the target analyte is accomplished by growing thetarget analyte in a nutrient medium so as to form colonies of the targetanalyte in the medium, and then detecting and counting any resultanttarget analyte colonies which may form in the medium.

The analysis is performed in a medium which may also contain, or iscombined with, one or more labeled analyte-specific materials (LASMs).The LASMs are homogeneously distributed throughout the medium The LASMsinclude a binding element, which may be a protein, lectin or anucleotide sequence that is specific to a binding site in or on thetarget analyte. The label may be a dye or a fluorophore, including acolloid or particle, which is attached to the binding element. The LASMsin the medium can diffuse and migrate through the medium to the targetanalyte colonies, and will bind to the individual organisms in thecolonies thereby differentially highlighting the colonies relative tothe remainder of the medium. The LASMs may either bind directly to thesurface of the organism, or to a product of the organism, such as itscapsular material or an internal molecular structure, so long as theLASM, being so bound, does not appreciably migrate away from the site.Because the binding of the LASMs locally depletes the LASM concentrationin the regions of the medium occupied by the target analyte colonies, a“halo” of lower LASM concentration will form around each target analytecolony, and at the center of each halo will be a high intensity peakcorresponding to the labeled colony. Any non-target organisms present inthe medium will not bind the LASMs and will show this characteristic.

A sample of a material to be analyzed is inoculated into the medium ordistributed on the medium. If a quantitative analysis is desired, theamount of the sample inoculum will be volumetrically measured. As theorganisms reproduce, colonies will form and will remain in a fixedlocation in or on the medium. The LASMs within the medium are sized soas to be able to migrate in or through the medium. Therefore, the LASMsin the region of the of the target analyte colonies are free to movethrough the medium and bind to the target organisms in the colonies, orto their products. This will cause the target organism colonies to showa higher label signal than surrounding areas in the medium. The reasonfor this is that the target organism colonies become LASM-enriched, andthe surrounding areas become LASM-depleted. The immediumtely surroundingareas in the medium, which abut the target organism colonies, will tendto emit a weakened label signal. The net result is the formation of“bright” spots surrounded by “dim” halos in the medium. In the eventthat there is no target analyte present in the sample, then the mediumwill retain a relatively even level of label signal emission, and willappear to be evenly “colored”.

The assay can be completed within one to two hours after inoculating themedium with the sample if a sensitive opto-mechanical photometric readeris used such as the one described in co-pending application U.S. Ser.No. 09/255,673, filed Feb. 23, 1999; or within about eighteen hours if avisual inspection is used. Since one will know the volume of the sampleinoculated into or spread onto the medium, one can quantify the amountof the target analyte per unit volume of the specimen sample by countingthe number of highlighted colonies In the medium. This enables the useof the invention to obtain a quantitative analysis for target analyte.It is desirable that a portion of the medium which contains the LASM beprotected against sample inoculation, thus allowing this portion of themedium to serve as a negative control.

Some samples, especially those from “dirty” sources, such asenvironmental samples, or those containing a great deal of othermaterials, may have a large variety of particles or other material thatmay have some level of non-specific signal, and where these non-specificsignals may be confused with those from the target analytes. Thisproblem can be overcome, however, by visually or photometrically takingsequential images from the same areas in the medium, which images areseparated from each other by suitable periods of time. Only thosecolonies formed from the target organism will increase in signalintensity by virtue of their continued growth. A comparison ofsuccessive images, preferably performed by the use of digital imageprocessing, can separate these areas of increasing intensity from thesurrounding medium. This method of kinetic analysis is the preferredmethod in all circumstances where a suitable photometric reading deviceis available because this technique provides the greatest sensitivity,the best selectivity, and the fastest time to completion of theanalysis.

In order to give the greatest signal/noise ratio, it is desirable thatthe medium be as thin as possible, i.e., about one millimeter or less,and preferably less than about 0.50 millimeter. The concentration of thelabel should be such that the signal from it can just be detected from anon-inoculated area of the medium. For a visual examination, a usefulsignal would be one that is slightly visible using the naked eye underthe appropriate lighting conditions and is not so marked so as toobscure the signal from the colonies. For automated examination, theconcentration should give an intensity which has an adequatesignal-to-noise ratio over a “dark” background which allows itsquantitation. The exact amount must be determined based upon thephotometric characteristics of the instrument used. Fluorophores such asfluorescein, sulforhodamine, and Cy-3, Cy-5 and Cy-7 can be used aslabels with the photometric analysis protocol. Note that two or moretarget organisms can be detected using this method if the labels of theLASMs for each target can be distinguished, for example, by color,and/or light wave emissions from the LASMs.

It is therefore an object of this invention to provide a method andapparatus which can be used to assay a specimen sample for the presenceor absence of a target analyte.

It is another object of this invention to provide a method and apparatusof the character described wherein the sample is placed on a mediumwhich is combined with a target analyte-specific labeled material thatcan migrate within the medium.

It is a further object of this invention to provide a method andapparatus of the character described wherein the target analyte is aliving organism.

It is another object of this invention to provide a method and apparatusof the character described which can detect two or more target analyteson the same medium.

It is an additional object of this invention to provide a method andapparatus of the character described wherein the target analyte isdetected by growing colonies of the target analyte in the medium, andthus creating increasingly localized concentrations of theanalyte-specific labeled material in the colonies of the target analyte.

These and other objects and advantages of the invention will become morereadily apparent from the following detailed description of theinvention when taken in conjunction with the accompanying drawings, inwhich:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a labeled target analyte-specific material andtarget analyte growth medium mixture formed in accordance with thisinvention;

FIGS. 2 and 3 are plan views similar to FIG. 1 but showing the formationof varying degrees of localized intensely labeled areas in the mixturedue to the presence of the target analyte in the sample being assayed;and

FIG. 4 is a pictograph trace of the signal intensity levels emanatingfrom the sample as the latter is scanned by a photometric light emissionsignal detection instrument, or of the visually observed signalintensity levels.

DETAILED EXAMPLE FOR CARRYING OUT THE INVENTION

Referring now to FIG. 1, there is shown a plan view of a rectangularsection of a medium, denoted generally by the numeral 2, which isadapted for performing the method of this invention. The medium field asshown in FIG. 1 is evenly shaded so as to indicate an evenly distributedlabel signal emission which will be detected by the human eye or by ascanning instrument either before the medium has been inoculated withthe sample; or after inoculation and incubation of the sample when thereis no target analyte present in the sample. The area 3 in the medium 2is an area which will not be inoculated with the sample being assayed,but which contains all of the reagents used in the sample, and thereforeserves as a negative control area. FIGS. 2 and 3 are illustrative of theresulting change in the label emission pattern in the medium 2 when thetarget analyte is present in the sample. FIG. 2 shows the result of arelatively low level of target analyte in the sample which will producea plurality of more highly labeled colonies 4 surrounded by halos 6 oflower level label in the medium 2. FIG. 3 shows the result when there isa relatively high level of target analyte in the sample. It will benoted from FIGS. 2 and 3 that one can count the colonies 4, and sincethe volume of the sample inoculum and the size of the field of medium 2can be known, one can derive a concentration of analyte per unit volumeof the sample by following the procedure of this invention. FIG. 4 is apictograph of the emission intensity from the label that will bedetected by an auto-analyzing instrument as it performs a linear scan ofthe medium. The trace 8 represents the emission levels from the label inthe medium; the dips 10 represent the low levels of label signalemission emanating from the “dim” halos 6; and the peak 12 representsthe high level of label signal emission emanating from the “bright”colonies 4.

The following are examples of target analytes which can be detectedutilizing the technique of this invention, and reagents for use indetecting each analyte.

EXAMPLE 1

In the case of the detection of E.coli 0157:H7 from a food orenvironmental source, a LASM is made by covalently binding afluorophore, such as fluorescein or Texas Red, to an antibody which isspecific to the 0157:H7 organism. The LASM is mixed with a growth mediumsuch as Muller-Hinton agar, MacConkey's Sorbital medium, or the like,such that there results a sufficient concentration of the LASM to eitherappear visible when illuminated by light of an appropriate wavelength,or be readily measurable by an imaging system. In the case of visualdetection, fluorescein is the preferred label. When a photometricanalysis protocol is used, the label can be a sulphorhodamine label, ora Cy3, Cy5, or a Cy7 label, the latter of which Cy labels emit signalsin the red, or near infrared, wavelengths, and are not detectable by thehuman eye, but which are photometrically detectable.

A plate is poured with a thickness of preferably 0.5 millimeter of themedium/LASM mixture for use in an instrumental detection format; or withabout 1.0 millimeter thick layer of the mixture for use in a visualdetection format. If the sample is from a clinical source, it may beplated directly on the medium. If the sample is from a food source, thefood source will preferably be dispersed in a sterile solution and thenfiltered to remove visible particulate materials from the sample.Samples from environmental sources can be treated in the same manner asfood samples. After the sample is incubated at a temperature of about35° C., the plated sample will be periodically examined as describedabove. Samples suspected of containing only a few organisms may beconcentrated by, for example, filtering a volume of material to betested and then placing the filter on the medium. Inasmuch as the filteris by definition permeable, the LASMs can pass through the filter andattach to the organisms growing thereon, as if the organisms weregrowing on the surface of the medium itself.

EXAMPLE 2

In the case of the detection of Listeria monocytogenes, such as from asample of cheese, the analysis is preferably performed in accordancewith Example 1 above, except that the LASM is made by covalently bindingthe fluorophore or other dye to an antibody specific to the Listeriaorganism. The LASM is mixed with a growth medium such as Esculin-basedgram positive selective agar. After the sample being analyzed isinoculated onto the medium, the plate is incubated at 35° C. The plateis then periodically visually or photometrically examined as describedabove.

EXAMPLE 3

In the case of the detection of Salmonella typhi, such as from a sampleof food, water, or a clinical source, the analysis is preferablyperformed in accordance with Example 1 above, except that the LASM ismade by covalently binding the fluorophore or other dye to an antibodyspecific to the Salmonella typhi organism. The LASM is mixed with agrowth medium such as Bismuth sulfite agar or Hektoen enteric agar.After the sample being analyzed is inoculated onto or into the medium,the plate is incubated at 35° C. The plate is then periodically visuallyor photometrically examined as described above.

EXAMPLE 4

In the case of the detection of the yeast Candida albicans from aclinical sample, the analysis is preferably performed in accordance withExample 1 above, except that the LASM is made by covalently binding thefluorophore or other dye to an antibody or lectin specific to theCandida albicans organism. The LASM is mixed with a growth medium suchas a Sabouraud's dextrous agar medium. After the sample being analyzedis inoculated onto the medium, the plate is incubated at 35° C. Theplate is then periodically visually or photometrically examined asdescribed above.

It will be readily appreciated that the above-described method andparaphernalia can be utilized to detect the presence or absence ofvarious microbes which may be present in a sample being analyzed. Thecreation of the localized dimly labeled halos which surround brightlylabeled target microbe colonies that may form in the growth mediumallows either visual or photometric detection, and counting of anyformed target microbe colonies in the growth medium When utilized, theselective nature of the growth medium will enhance target microbe colonygrowth and suppress non-target microbe colony growth. The technique canbe used to assess the concentration of target microbes in the sample bysimply counting the formed colonies and recording the volume of samplewhich is inoculated onto or into the growth medium. Sequential readingscan be taken from identical areas of the medium to detect changes inlocal LASM concentration, which changes are related to target analytegrowth.

Since many changes and variations of the disclosed embodiments of theinvention may be made without departing from the inventive concept, itis not intended to limit the invention otherwise than as required by theappended claims.

We claim:
 1. An assembly for use in visually or photometricallydetecting one or more live target microbial organism analyte(s) in asample of a material which is suspected to contain the targetanalyte(s), said assembly comprising: a) a sample container; b) agrowth-supporting medium in said sample container, said medium beingoperative to support metabolic replication of said target analyte(s) soas to enable said target analyte(s) to form target analyte colonies inor on said medium, and said medium being a medium which restrictsintra-medium migration of target analyte colonies which form in or onsaid medium; and c) one or more labeled target analyte-specificmaterial(s) (LASM(s)) which are homogeneously dispersed throughout saidgrowth-supporting medium and which LASM(s) migrate within saidgrowth-supporting medium toward target analyte colonies which form in oron said growth-supporting medium so as to differentially highlight saidtarget analyte colonies.
 2. The assembly of claim 1 wherein saidgrowth-supporting medium is a medium which promotes metabolic targetanalyte colony formation and which inhibits non-target analyte metaboliccolony formation in or on said growth-supporting medium.
 3. The assemblyof claim 1 wherein said target analyte(s) include E.coli 0157:H7; saidLASM(s) include an E.coli 01 57:H7-specific antibody coupled with adetectable label; and said growth-supporting medium is agrowth-supporting medium selected from the group consisting of aMuller-Hinton agar Kirby-Bauer medium; and a MacConkey's Sorbitalmedium.
 4. The assembly of claim 3 wherein said label is a labelselected from the group consisting of fluorophores, Cy-3, Cy-5, andCy-7.
 5. The assembly of claim 4 wherein said label is a sulforhodamine.6. The assembly of claim 4 wherein said label is a fluorescein.
 7. Theassembly of claim 1 wherein said target analyte(s) include Listeriamonocytogenes; said LASM(s) include a Listeria monocytogenes-specificantibody coupled with a detectable label; and said growth-supportingmedium is Esculin-based gram positive selective agar.
 8. The assembly ofclaim 7 wherein said label is a fluorophore.
 9. The assembly of claim 8wherein said fluorophore is a sulforhodamine.
 10. The assembly of claim8 wherein said fluorophore is a fluorescein.
 11. The assembly of claim 1wherein said target analyte(s) include Salmonella typhosa; said LASM(s)include a Salmonella typhi-specific antibody coupled with a detectablelabel; and said growth-supporting medium is an agar medium selected fromthe group consisting of Bismuth sulfite agar and Hektoen enteric agar.12. The assembly of claim 11 wherein said label is a fluorophore. 13.The assembly of claim 11 wherein said fluorophore a sulforhodamine. 14.The assembly of claim 11 wherein said fluorophore is a fluorescein. 15.The assembly of claim 1 wherein said target analyte(s) include Candidaalbicans, said LASM(s) include a Candida albicans-specific antibody orlectin which is combined with a detectable label; and saidgrowth-supporting medium is a Muller-Hinton agar Kirby-Bauer medium. 16.The assembly of claim 15 wherein said label is a fluorophore.
 17. Theassembly of claim 16 wherein said fluorophore is a sulforhodamine. 18.The assembly of claim 16 wherein said fluorophore is a fluorescein. 19.An assembly for use in photometrically detecting one or more live targetmicrobial organism analyte(s) in a sample of a material which issuspected to contain the target analyte(s), said assembly comprising: a)a sample container; b) a growth-supporting medium in said samplecontainer, said growth-supporting medium being operative to supportmetabolic replication of said target analyte(s) so as to enable saidtarget analyte(s) to form target analyte colonies in or on said medium,and said medium being a medium which restricts intra-medium migration oftarget microbial analyte colonies which form in or on said medium; andc) one or more labeled analyte-specific material(s) (LASM(s)) which arehomogeneously dispersed throughout said growth-supporting medium andwhich LASM(s) migrate within said growth-supporting medium toward targetanalyte colonies which form in or on said growth-supporting medium so asto differentially highlight said target analyte colonies.
 20. Theassembly of claim 19 wherein said growth-supporting medium is a mediumwhich promotes metabolic target analyte colony formation and whichinhibits metabolic non-target analyte microbial colony formation in oron said growth-supporting medium.
 21. The assembly of claim 19 whereinsaid LASM(s) include a photometrically detectable label selected fromthe group consisting of Cy-3, Cy-5, and Cy-7.
 22. A method forphotometrically detecting one or more live target microbial analyte(s)in a sample of a material which is suspected to contain the targetanalyte(s), said method comprising: a) the step of providing a samplecontainer; b) the step of providing a growth-supporting medium in saidsample container, said growth-supporting medium being a medium which isoperative to support metabolic replication of said target analyte(s) soas to enable said target analyte(s) to form target analyte colonies inor on said medium, and said being a medium which restricts intra-mediummigration of target microbial analyte colonies which form in or on saidmedium; c) the step of providing one or more labeled analyte-specificmaterial(s) (LASM(s)) which are homogeneously dispersed throughout saidgrowth-supporting medium, said LASM(s) being photometrically detectableand which LASM(s) migrate within said growth-supporting medium towardtarget analyte colonies which form in or on said growth-supportingmedium; d) the step of inoculating the material(s) being analyzed ontoor into said growth-supporting medium; and e) the step ofphotometrically scanning the inoculated medium for the presence orabsence of brightly labeled target analyte colonies.
 23. The method ofclaim 22 wherein said growth-supporting medium is a medium whichpromotes metabolic target analyte colony formation and which inhibitsmetabolic non-target analyte colony formation in or on saidgrowth-supporting medium.
 24. The method of claim 22 wherein saidLASM(s) include a photometrically detectable label selected from thegroup consisting of Cy-3, Cy-5, and Cy-7.
 25. The method of claim 22wherein said brightly labeled target analyte colonies are surrounded bydimly labeled halos in said medium.
 26. The method of claim 22 whereinsequential readings are taken from identical areas of the medium todetect changes in local LASM concentration which changes are related tometabolic target analyte growth.